Animal cells and processes for the replication of influenza viruses

ABSTRACT

Animal cells are described which can be infected by influenza viruses and which are adapted to growth in suspension in serum-free medium. Processes for the replication of influenza viruses in cell culture using these cells are furthermore described, as well as vaccines which contain the influenza viruses obtainable by the process or constituents thereof.

This application is a continuation of U.S. Ser. No. 11/057,370, filedFeb. 15, 2005, which is a continuation of U.S. Ser. No. 10/670,788,filed Sep. 26, 2003, now abandoned, which is a continuation of U.S. Ser.No. 10/194,784, filed Jul. 12, 2002, now U.S. Pat. No. 6,656,720, whichis a continuation of application U.S. Ser. No. 09/155,581, filed Sep.29, 1998, now U.S. Pat. No. 6,455,298, which is a U.S. National Stageapplication under 35 U.S.C. § 371 of International Application No.PCT/IB97/00403, filed Apr. 1, 1997, which claims priority to GermanApplication No. 196 12 966.4, filed Apr. 1, 1996. The disclosure of eachof the foregoing is hereby incorporated by reference in its entirety.

The present invention relates to animal cells which can be infected byinfluenza viruses and are adapted to growth in suspension in serum-freemedium, and to processes for the replication of influenza viruses incell culture using these cells. The present invention further relates tothe influenza viruses obtainable by the process described and tovaccines which contain viruses of this type or constituents thereof.

All influenza vaccines which have been used since the 40s until today aspermitted vaccines for the treatment of humans and animals consist ofone or more virus strains which have been replicated in embryonate hens'eggs. These viruses are isolated from the allantoic fluid of infectedhens' eggs and their antigens are used as vaccine either as intact virusparticles or as virus particles disintegrated by detergents and/orsolvents—so-called cleaved vaccine—or as isolated, defined virusproteins—so-called subunit vaccine. In all permitted vaccines, theviruses are inactivated by processes known to the person skilled in theart. The replication of live attenuated viruses, which are tested inexperimental vaccines, is also carried out in embryonate hens' eggs.

The use of embryonate hens' eggs for vaccine production is time-, labor-and cost-intensive. The eggs—from healthy flocks of hens monitored byveterinarians—have to be incubated before infection, customarily for 12days. Before infection, the eggs have to be selected with respect toliving embryos, as only these eggs are suitable for virus replication.After infection the eggs are again incubated, customarily for 2 to 3days. The embryos still alive at this time are killed by cold and theallantoic fluid is then obtained from the individual eggs by aspiration.By means of laborious purification processes, substances from the hen'segg which lead to undesired side effects of the vaccine are separatedfrom the viruses, and the viruses are concentrated. As eggs are notsterile (pathogen-free), it is additionally necessary to remove and/orto inactivate pyrogens and all pathogens which are possibly present.

Viruses of other vaccines such as, for example, rabies viruses, mumps,measles, rubella, polio viruses and FSME viruses can be replicated incell cultures. As cell cultures originating from tested cell banks arepathogen-free and, in contrast to hens' eggs, are a defined virusreplication system which (theoretically) is available in almostunlimited amounts, they make possible economical virus replication undercertain circumstances even in the case of influenza viruses. Economicalvaccine production is possibly also achieved in that virus isolation andpurification from a defined, sterile cell medium appears simpler thanfrom the strongly protein-containing allantoic fluid. The isolation andreplication of influenza viruses in eggs leads to a selection of certainphenotypes, of which the majority differ from the clinical isolate. Incontrast to this is the isolation and replication of the viruses in cellculture, in which no passage-dependent selection occurs (Oxford, J. S.et al., J. Gen. Virology 72 (1991), 185-189; Robertson, J. S. et al., J.Gen. Virology 74 (1993)2047-2051). For an effective vaccine, therefore,virus replication in cell culture is also to be preferred from thisaspect to that in eggs.

It is known that influenza viruses can be replicated in cell cultures.Beside hens' embryo cells and hamster cells (BHK21-F and HKCC), MDBKcells, and in particular MDCK cells have been described as suitablecells for the in-vitro replication of influenza viruses (Kilbourne, E.D., in: Influenza, pages 89-110, Plenum Medical Book Company—New Yorkand London, 1987). A prerequisite for a successful infection is theaddition of proteases to the infection medium, preferably trypsin orsimilar serine proteases, as these proteases extracellularly cleave theprecursor protein of hemagglutinin [HA₀] into active hemagglutinin [HA₁and HA₂]. Only cleaved hemagglutinin leads to the adsorption of theinfluenza viruses on cells with subsequent virus assimilation into thecells (Tobita, K. et al., Med Microbiol. Immunol., 162 (1975), 9-14;Lazarowitz, S. G. & Choppin, P. W., Virology, 68 (1975) 440-454; Klenk,H.-D. et al., Virology 68 (1975) 426-439) and thus to a furtherreplication cycle of the virus in the cell culture.

The U.S. Pat. No. 4,500,513 described the replication of influenzaviruses in cell cultures of adherently growing cells. After cellproliferation, the nutrient medium is removed and fresh nutrient mediumis added to the cells with infection of the cells with influenza virusestaking place simultaneously or shortly thereafter. A given time afterthe infection, protease (e.g. trypsin) is added in order to obtain anoptimum virus replication. The viruses are harvested, purified andprocessed to give inactivated or attenuated vaccine. Economicalinfluenza virus replication as a prerequisite for vaccine productioncannot be accomplished, however, using the methodology described in thepatent mentioned, as the change of media, the subsequent infection aswell as the addition of trypsin which is carried out later necessitatesopening the individual cell culture vessels several times and is thusvery labor-intensive. Furthermore, the danger increases of contaminationof the cell culture by undesirable micro-organisms and viruses with eachmanipulation of the culture vessels. A more cost-effective alternativeis cell proliferation in fermenter systems known to the person skilledin the art, the cells growing adherently on microcarriers. The serumnecessary for the growth of the cells on the microcarriers (customarilyfetal calf serum), however, contains trypsin inhibitors, so that even inthis production method a change of medium to serum-free medium isnecessary in order to achieve the cleavage of the influenzahemagglutinin by trypsin and thus an adequately high virus replication.Thus this methodology also requires opening of the culture vesselsseveral times and thus brings with it the increased danger ofcontamination.

The present invention is thus based on the object of making availablecells and processes which make possible simple and economical influenzavirus replication in cell culture. This object is achieved by theprovision of the embodiments indicated in the patent claims. Theinvention thus relates to animal cells which can be infected byinfluenza viruses and which are adapted to growth in suspension inserum-free medium. It was found that it is possible with the aid ofcells of this type to replicate influenza viruses in cell culture in asimple and economical manner. By the use of the cells according to theinvention, on the one hand a change of medium before infection to removeserum can be dispensed with an on the other hand the addition ofprotease can be carried out simultaneously to the infection. On thewhole, only a single opening of the culture vessel for infection withinfluenza viruses is thus necessary, whereby the danger of thecontamination of the cell cultures is drastically reduced. Theexpenditure of effort which would be associated with the change ofmedium, the infection and the subsequent protease addition isfurthermore decreased. A further advantage is that the consumption ofmedia is markedly decreased.

The cells according to the invention are preferably vertebrate cells,e.g. avian cells, in particular hens' embryo cells. In a particularlypreferred embodiment, the cells according to the invention are mammaliancells, e.g. from hamsters, cattle, monkeys or dogs, in particular kidneycells or cell lines derived from these. They are preferably cells whichare derived from MDCK cells (ATCC CCL34 MDCK (NBL-2)), and particularlypreferably cells of the cell line MDCK 33016. This cell line wasdeposited under the deposit number DSM ACC2219 on Jun. 7, 1995 accordingto the requirements of the Budapest Convention for the InternationalRecognition of the Deposition of Micro-organisms for the Purposes ofPatenting in the German Collection of Micro-organisms (DSM), inBrunswick, Federal Republic of Germany, recognized as the internationaldeposition site. The cell line MDCK 33016 is derived from the cell lineMDCK by passaging and selection with respect to the capability ofgrowing in suspension in serum-free medium and of replicating variousviruses, e.g. orthomyxoviruses, paramyxoviruses, rhabdoviruses andflavoviruses. On account of these properties, these cells are suitablefor economical replication of influenza viruses in cell culture by meansof a simple and cost-effective process.

The present invention therefore also relates to a process for thereplication of influenza viruses in cell culture, in which cellsaccording to the invention are used, in particular a process whichcomprises the following steps:

i) proliferation of the cells according to the invention described abovein serum-free medium in suspension;

ii) infection of the cells with influenza viruses;

iii) addition of protease shortly before, simultaneously to or shortlyafter infection; and

iv) further culturing of the infected cells and isolation of thereplicated influenza viruses.

The cells according to the invention can be cultured in the course ofthe process in various serum-free media known to the person skilled inthe art (e.g. Iscove's medium, ultra CHO medium (BioWhittaker), EX-CELL(JRH Biosciences)). Otherwise, the cells for replication can also becultured in the customary serum-containing media (e.g. MEM or DMEMmedium with 0.5% to 10%, preferably 1.5% to 5%, of fetal calf serum) orprotein-free media (e.g. PF-CHO (JRH Biosciences)). Suitable culturevessels which can be employed in the course of the process according tothe invention are all vessels known to the person skilled in the art,such as, for example, spinner bottles, roller bottles or fermenters.

The temperature for the proliferation of the cells before infection withinfluenza viruses is preferably 37° C. Culturing for proliferation ofthe cells (step (i)) is carried out in a preferred embodiment of theprocess in a perfusion system, e.g. in a stirred vessel fermenter, usingcell retention systems known to the person skilled in the art, such as,for example, centrifugation, filtration, spin filters and the like.

The cells are in this case preferably proliferated for 2 to 18 days,particularly preferably for 3 to 11 days. Exchange of the medium iscarried out in the course of this, increasing from 0 to approximately 1to 3 fermenter volumes per day. The cells are proliferated up to veryhigh cell densities in this manner, preferably up to approximately 2×10⁷cells/ml. The perfusion rates during culture in the perfusion system canbe regulated both via the cell count, the content of glucose, glutamineor lactate in the medium and via other parameters known to the personskilled in the art. For infection with influenza viruses, about 85% to99%, preferably 93 to 97%, of the fermenter volume is transferred withcells to a further fermenter. The cells remaining in the first fermentercan in turn be mixed with medium and replicated further in the perfusionsystem. In this manner, continuous cell culture for virus replication isavailable.

Alternatively to the perfusion system, the cells in step (i) of theprocess according to the invention can preferably also be cultured in abatch process. The cells according to the invention proliferate here at37° C. with a generation time of 20 to 30 h up to a cell density ofabout 8 to 25×10⁵ cells/ml.

In a preferred embodiment of the process according to the invention, thepH of the culture medium used in step (i) is regulated during culturingand is in the range from pH 6.6 to pH 7.8, preferably in the range frompH 6.8 to pH 7.3.

Furthermore, the pO₂ value is advantageously regulated in this step ofthe process and is preferably between 25% and 95%, in particular between35% and 60% (based on the air saturation). According to the invention,the infection of the cells cultured in suspension is preferably carriedout when the cells in the batch process have achieved a cell density ofabout 8 to 25×10⁵ cells/ml or about 5 to 20×10⁶ cells/ml in theperfusion system.

In a further preferred embodiment, the infection of the cells withinfluenza viruses is carried out at an m.o.i. (multiplicity ofinfection) of about 0.0001 to 10, preferably of 0.002 to 0.5. Theaddition of the protease which brings about the cleavage of theprecursor protein of hemagglutinin [HA₀] and thus the adsorption of theviruses on the cells, can be carried out according to the inventionshortly before, simultaneously to or shortly after the infection of thecells with influenza viruses. If the addition is carried outsimultaneously to the infection, the protease can either be addeddirectly to the cell culture to be infected or, for example, as aconcentrate together with the virus inoculate. The protease ispreferably a serine protease, and particularly preferably trypsin.

In a preferred embodiment, trypsin is added to the cell culture to beinfected up to a final concentration of 1 to 200 μg/ml, preferably 5 to50 μg/ml, and particularly preferably 5 to 30 μg/ml in the culturemedium. During the further culturing of the infected cells according tostep (iv) of the process according to the invention, trypsinreactivation can be carried out by fresh addition of trypsin in the caseof the batch process or in the case of the perfusion system bycontinuous addition of a trypsin solution or by intermittent addition.In the latter case, the trypsin concentration is preferably in the rangefrom 1 μg/ml to 80 μg/ml.

After infection, the infected cell culture is cultured further toreplicate the viruses, in particular until a maximum cytopathic effector a maximum amount of virus antigen can be detected. Preferably, theculturing of the cells is carried out for 2 to 10 days, in particularfor 3 to 7 days. The culturing can in turn preferably be carried out inthe perfusion system or in the batch process.

In a further preferred embodiment, the cells are cultured at atemperature of 30° C. to 36° C., preferably of 32° C. to 34° C., afterinfection with influenza viruses. The culturing of the infected cells attemperatures below 37° C., in particular in the temperature rangesindicated above, leads to the production of influenza viruses whichafter inactivation have an appreciably higher activity as vaccine, incomparison with influenza viruses which have been replicated at 37° C.in cell culture.

The culturing of the cells after infection with influenza viruses (step(iv)) is in turn preferably carried out at regulated pH and pO₂. The pHin this case is preferably in the range from 6.6 to 7.8, particularlypreferably from 6.8 to 7.2, and the pO₂ in the range from 25% to 150%,preferably from 30% to 75%, and particularly preferably in the rangefrom 35% to 60% (based on the air saturation).

During the culturing of the cells or virus replication according to step(iv) of the process, a substitution of the cell culture medium withfreshly prepared medium, medium concentrate or with defined constituentssuch as amino acids, vitamins, lipid fractions, phosphates etc. foroptimizing the antigen yield is also possible.

After infection with influenza viruses, the cells can either be slowlydiluted by further addition of medium or medium concentrate over severaldays or can be incubated during further perfusion with medium or mediumconcentrate decreasing from approximately 1 to 3 to 0 fermentervolumes/day. The perfusion rates can in this case in turn be regulatedby means of the cell count, the content of glucose, glutamine, lactateor lactate dehydrogenase in the medium or other parameters known to theperson skilled in the art.

A combination of the perfusion system with a fed-batch process isfurther possible. In a preferred embodiment of the process, theharvesting and isolation of the replicated influenza viruses is carriedout 2 to 10 days, preferably 3 to 7 days, after infection. To do this,for example, the cells or cell residues are separated from the culturemedium by means of methods known to the person skilled in the art, forexample by separators or filters. Following this the concentration ofthe influenza viruses present in the culture medium is carried out bymethods known to the person skilled in the art, such as, for example,gradient centrifugation, filtration, precipitation and the like.

The invention further relates to influenza viruses which are obtainableby a process according to the invention. These can be formulated byknown methods to give a vaccine for administration to humans or animals.The immunogenicity or efficacy of the influenza viruses obtained asvaccine can be determined by methods known to the person skilled in theart, e.g. by means of the protection imparted in the loading experimentor as antibody titers of neutralizing antibodies. The determination ofthe amount of virus or antigen produced can be carried out, for example,by the determination of the amount of hemagglutinin according to methodsknown to the person skilled in the art. It is known, for example, thatcleaved hemagglutinin binds to erythrocytes of various species, e.g. tohens' erythrocytes. This makes possible a simple and rapidquantification of the viruses produced or of the antigen formed.

Thus the invention also relates to vaccines which contain influenzaviruses obtainable from the process according to the invention. Vaccinesof this type can optionally contain the additives customary forvaccines, in particular substances which increase the immune response,i.e. so-called adjuvants, e.g. hydroxide of various metals, constituentsof bacterial cell walls, oils or saponins, and moreover customarypharmaceutically tolerable excipients.

The viruses can be present in the vaccines as intact virus particles, inparticular as live attenuated viruses. For this purpose, virusconcentrates are adjusted to the desired titer and either lyophilized orstabilized in liquid form.

In a further embodiment, the vaccines according to the invention cancontain disintegrated, i.e. inactivated, or intact, but inactivatedviruses. For this purpose, the infectiousness of the viruses isdestroyed by means of chemical and/or physical methods (e.g. bydetergents or formaldehyde). The vaccine is then adjusted to the desiredamount of antigen and after possible admixture of adjuvants or afterpossible vaccine formulation, dispensed, for example, as liposomes,microspheres or “slow release” formulations.

In a further preferred embodiment, the vaccines according to theinvention can finally be present as subunit vaccine, i.e. they cancontain defined, isolated virus constituents, preferably isolatedproteins of the influenza virus. These constituents can be isolated fromthe influenza viruses by methods known to the person skilled in the art.

Furthermore, the influenza viruses obtained by the process according tothe invention can be used for diagnostic purposes. Thus the presentinvention also relates to diagnostic compositions which containinfluenza viruses according to the invention or constituents of suchviruses, if appropriate in combination with additives customary in thisfield and suitable detection agents. The examples illustrate theinvention.

EXAMPLE 1 Preparation of Cell Lines which are Adapted to Growth inSuspension and can be Infected by Influenza Viruses

A cell line which is adapted to growth in suspension culture and can beinfected by influenza viruses is selected starting from MDCK cells (ATCCCCL34 MDCK (NBL-2), which had been proliferated by means of only a fewpassages or over several months in the laboratory. This selection wascarried out by proliferation of the cells in roller bottles which wererotated at 16 rpm (instead of about 3 rpm as is customary for rollerbottles having adherently growing cells). After several passages of thecells present suspended in the medium, cell strains growing insuspension were obtained. These cell strains were infected withinfluenza viruses and the strains were selected which produced thehighest virus yield. An increase in the rate of cells growing insuspension during the first passages at 16 rpm is achieved over 1 to 3passages by the addition of selection systems known to the personskilled in the art, such as hypoxanthine, aminopterin and thymidine, oralanosine and adenine, individually or in combination. The selection ofcells growing in suspension is also possible in other agitated cellculture systems known to the person skilled in the art, such as stirredflasks.

Alternatively, highly virus-replicating cell clones can be establishedbefore selection as suspension cells by cell cloning in microtiterplates. In this process, the adherently growing starting cells (aftertrypsinization) are diluted to a concentration of about 25 cells/ml withserum-containing medium and 100 μl each of this cell suspension areadded to a well of a microtiter plate. If 100 μl of sterile-filteredmedium from a 2 to 4-day old (homologous) cell culture (“conditionedmedium”) are added to each well, the probability of growth of the cellsinoculated at a very low cell density increases. By means oflight-microscopic checking, the wells are selected in which only onecell is contained; the cell lawn resulting therefrom is then passaged inlarger cell culture vessels. The addition of selection media (e.g.hypoxanthine, aminopterin and thymidine, or alanosine and adenine,individually or in combination) after the 1st cell passage leads over 1to 3 passages to a greater distinguishability of the cell clones. Thecell clones resulting in this way were selected with respect to theirspecific virus replication and then selected as suspension cells. Theselection of cells which are adapted to growth in serum-free medium canalso be carried out by methods known to the person skilled in the art.

Examples of cells which are adapted to growth in serum-free medium insuspension and can be infected by influenza viruses are the cell linesMDCK 33016 (DSM ACC2219) and MDCK 13016, whose properties are describedin the following examples.

EXAMPLE 2 Replication of Influenza Viruses in the Cell Line MDCK 33016

The cell line MDCK 33016 (DSM ACC2219; obtained from an MDCK cellculture by selection pressure) was proliferated at 37° C. in Iscove'smedium with a splitting rate of 1:8 to 1:12 twice weekly in a rollerbottle which rotated at 16 rpm. Four days after transfer, a cell countof approximately 7.0×10⁵ to 10×10⁵ cells/ml was achieved. Simultaneouslyto the infection of the now 4-day old cell culture with the influenzavirus strain A/PR/8/34 (m.o.i.=0.1), the cell culture was treated withtrypsin (25 μg/ml final concentration) and cultured further at 37° C.,and the virus replication was determined over 3 days (Table I).

TABLE I Replication of influenza A/PR/8/34 in roller bottles (cell lineMDCK 33016) after infection of a cell culture without change of medium,measured as antigen content (HA units) HA content after days afterinfection (dpi) 1 dpi 2 dpi 3 dpi Experiment 1 1:64 1:512 1:1024Experiment 2 1:4 1:128 1:1024 Experiment 3 1:8 1:32 1:512

The ratios indicated mean that a 1:X dilution of the virus harvest stillhas hemagglutinating properties. The hemagglutinating properties can bedetermined, for example, as described in Mayer et al., VirologischeArbeitsmethoden, [Virological Working Methods], Volume 1 (1974), pages260-261 or in Grist, Diagnostic Methods in Clinical Virology, pages 72to 75.

EXAMPLE 3 Replication of Influenza Viruses in the Cell Line MDCK 13016in Spinner Bottles

The cell line MDCK 13016 was replicated at 37° C. in Iscove's mediumwith a splitting rate of 1:6 to 1:10 twice weekly in a spinner bottle(50 rpm). Four days after transfer, a cell count of 8.0×10⁵ cells/ml wasachieved. Simultaneously to the infection of the now 4 day old cellculture with the influenza virus strain A/PR/8/34 (m.o.i.=0.1), the cellculture was treated with trypsin (25 μg/ml final concentration) andincubated further at 33° C. and the virus replication was determinedover 6 days (Table II).

TABLE II Replication of influenza A/PR/8/34 in spinner bottles (cellline MDCK 13016) after infection of a cell culture without change ofmedium, measured as antigen content (HA units) HA content after daysafter infection (dpi) 1 dpi 3 dpi 4 dpi 5 dpi 6 dpi Experiment 1 1:21:128 1:1024 1:1024 1:2048 Experiment 2 1:4 1:512 1:2048 1:2048 1:1024

EXAMPLE 4 Replication of Various Influenza Strains in the Cell Line MDCK33016 in Roller Bottles

The cell line MDCK 33016 (DSM ACC2219) was replicated at 37° C. inIscove's medium with a splitting rate of 1:8 to 1:12 twice weekly in aroller bottle which rotated at 16 rpm. Four days after transfer, a cellcount of approximately 7.0×10⁵ to 10×10⁵ cells/ml was achieved.Simultaneously to the infection of the now 4-day old cell culture withvarious influenza virus strains (m.o.i. 0.1), the cell culture wastreated with trypsin (25 μg/ml final concentration) and furtherincubated at 33° C., and the virus replication was determined on the 5thday after infection (Table III).

TABLE III Replication of influenza strains in roller bottles (cell lineMDCK 33016) after infection of a cell culture without change of medium,measured as antigen content (HA units) HA content 5 days after infectionInfluenza strain HA content A/Singapore/6/86 1:1024 A/Sichuan/2/87 1:256A/Shanghai/11/87 1:256 A/Guizhou/54/89 1:128 A/Beijing/353/89 1:512B/Beijing/1/87 1:256 B/Yamagata/16/88 1:512 A/PR/8/34 1:1024 A/Equi1/Prague 1:512 A/Equi 2/Miami 1:256 A/Equi 2 Fontainebleau 1:128A/Swine/Ghent 1:512 A/Swine/Iowa 1:1024 A/Swine/Amsberg 1:512

EXAMPLE 5 Replication of Various Influenza Strains in MDCK 33016 Cellsin the Fermenter

The cell line MDCK 33016 (DSM ACC2219) was inoculated in Iscove's mediumwith a cell inoculate of 1×10⁵ cells/ml in a stirred vessel fermenter(working volume 8 l). At an incubation temperature of 37° C., a pO₂ of50±10% (regulated) and a pH of 7.1±0.2 (regulated), the cellsproliferated within 4 days to a cell density of 7×10⁵ cells/ml. 8 ml ofvirus stock solution (either A/PR/8/34 or A/Singapore/6/86 orA/Shanghai/11/87 or A/Beijing/1/87 or B/Massachusetts/71 orB/Yamagata/16/88 or B/Panama/45/90) and simultaneously 16 ml of a 1.25%strength trypsin solution were added to these cells and the inoculatedcell culture was incubated further at 33° C. The virus replication wasdetermined over 6 days (Table IV).

TABLE IV Replication of influenza virus strains in the fermenter (cellline MDCK 33016) after infection of a cell culture without change ofmedium, measured as antigen content (HA units) HA content after daysafter infection (dpi) 1 dpi 3 dpi 4 dpi 5 dpi 6 dpi A/PR/8/34 1:64 1:5121:1024 1:2048 1:2048 A/Singapore 1:32 1:512 1:2048 1:2048 1:1024A/Shanghai 1:8 1:128 1:256 1:256 1:512 A/Beijing 1:16 1:256 1:10241:1024 n.d. B/Yamagata 1:8 1:128 1:512 1:512 n.d. B/Massachusetts 1:41:128 1:256 1:512 n.d. B/Panama n.d. 1:128 1:256 n.d. 1:1024

EXAMPLE 6 Influence of the Infection Dose (m.o.i.) on Virus Replication

The cell line MDCK 13016 (obtained from an MDCK cell culture byselection pressure) was proliferated at 37° C. in ultra CHO medium witha splitting rate of 1:8 to 1:12 twice weekly in a roller bottle whichrotated at 16 rpm. Four days after transfer, a cell count ofapproximately 7.0×10⁵ to 10×10⁵ cells/ml was achieved. The influence ofthe infective dose (m.o.i.) on the yield of antigen and infectiousnesswas investigated. Simultaneously to the infection of the now 4 day-oldcell culture with the influenza virus stain A/PR/8/34 (m.o.i.=0.5 andm.o.i.=0.005), the cell culture was treated with trypsin (25 μg/ml finalconcentration) and incubated further at 37° C., and the virusreplication was determined over 3 days (Table V).

TABLE V Replication of influenza virus strain PR/8/34 in the cell lineMDCK 13016 in roller bottles after infection with an m.o.i. of 0.5 or0.005. The assessment of virus replication was carried out by antigendetection (HA) and infectiousness titer (CCID₅₀ cell culture infectivedose 50% in log₁₀) Days after infection 2 3 4 5 PR/8/34 HA CCID₅₀ HACCID₅₀ HA CCID₅₀ HA CCID₅₀ m.o.i. = 0.5 128 5.1 256 5.7 512 5.3 1024 5.4m.o.i. = 0.005 64 4.9 512 8.0 512 8.3 1024 8.3

The determination of the CCID₅₀ can in this case be carried out, forexample, according to the method which is described in Paul, Zell-undGewebekultur [Cell and tissue culture](1980), p. 395.

EXAMPLE 7 Influence of Media Substitution on Virus Replication

The cell line MDCK 33016 (DSM ACC2219) was proliferated at 37° C. inIscove's medium with a splitting rate of 1:8 to 1:12 twice weekly in aroller bottle which rotated at 16 rpm. Four days after transfer, a cellcount of approximately 7.0×10⁵ to 10×10⁵ cells/ml was achieved. Theinfluence of a media substitution on the yield of antigen andinfectiousness was investigated. The now 4-day old cell culture wasinfected with the influenza virus strain A/PR/8/34 (m.o.i.=0.05), thetrypsin addition (20 μg/ml final concentration in the roller bottle)being carried out by mixing the virus inoculum with the trypsin stocksolution. The cell culture was treated with additions of media andincubated further at 33° C., and the virus replication was determinedover 5 days (Table VI).

TABLE VI Replication of influenza A/PR/8/34 in roller bottles (cell lineMDCK 33016); addition of 5% (final concentration) of atriple-concentrated Iscove's medium, of glucose (final concentration 3%)or glucose and casein hydrolysate (final concentration 3% or 0.1%)measured as antigen content (HA units) HA content after days afterinfection (dpi) Addition 1 dpi 3 dpi 4 dpi 5 dpi — 1:16 1:256 1:10241:1024 (control) 3x Iscove's 1:8 1:128 1:1024 1:2048 Glucose 1:32 1:5121:2048 1:2048 Glucose/casein 1:8 1:128 1:512 1:1024 hydrolysate

EXAMPLE 8 Replication of Influenza Viruses in MDCK 33016 Cells in theFermenter and Obtainment of the Viruses

The cell line MDCK 33016 (ACC2219) was inoculated in Iscove's mediumwith a cell inoculate of 0.5×10⁵ cells/ml in a stirred vessel fermenter(working volume 10 l). At an incubation temperature of 37° C., a pO₂ of55±10% (regulated) and a pH of 7.1±0.2 (regulated), the cellsproliferated within 4 days to a cell density of 7×10⁵ cells/ml. 0.1 mlof virus stock solution (A/Singapore/6/86; m.o.i. about 0.0015) andsimultaneously 16 ml of a 1.25% strength trypsin solution were added tothese cells and the inoculated cell culture was incubated further at 33°C. The virus replication was determined after 5 days and the virus washarvested. Cells and cell residues were removed by tangential flowfiltration (Sarcoton Mini-Microsart Module with 0.45 μm pore size;filtration procedure according to the instructions of the manufacturer),no loss of antigen (measured as HA) being detectable in the filtrate.The virus material was concentrated from 9.5 l to 600 ml by freshtangential flow filtration (Sartocon Mini-Ultrasart Module with 100,000NMWS (nominal molecular weight separation limit); filtration procedureaccording to the instructions of the manufacturer). The amount ofantigen in the concentrate was 5120 HA units (start 256 HA units;concentration factor 20), while the infectiousness in the concentratewas 9.2 log₁₀ CCID₅₀ (start 8.9 log₁₀ CCID₅₀; concentration factor 16);the loss of antigen and infectiousness was less than 1%, measured in thefiltrate after the 100,000 NMWS filtration.

EXAMPLE 9 Replication of the Influenza Viruses in MDCK 33016 Cells inthe Perfusion Fermenter

1.6×10⁸ cells of the cell line MDCK 33016 (DSM ACC2219) were suspendedin UltraCHO medium (0.8×10⁵ cells/ml) in the reactor vessel of BiostatMD (Braun Biotech Int., Melsungen, Germany) with an effective volume of2000 ml and proliferated at 37° C. in perfusion operation with a risingflow rate (entry of oxygen by hose aeration (oxygen regulation 40±10%pO₂); pH regulation pH £7.2; cell retention by spin filter >95%). Thelive cell count increased within 11 days by 200-fold to 175×10⁵ cells/ml(Table VIIIa). 1990 ml of this cell culture were transferred to a 2ndperfusion fermenter (working volume 5 l), while the remaining cells weremade up to 2000 ml again with medium and cell proliferation was carriedout again in perfusion operation. In the 2nd perfusion fermenter (virusinfection), the cells were infected with the influenza virus strainA/PR/8/34 (m.o.i.=0.01) with simultaneous addition of trypsin (10 μg/mlfinal concentration) and incubated for 1 h. The fermenter was thenincubated further in perfusion operation (regulation of pO₂: 40±10% andpH: £7.2). On the first day after infection, incubation was carried outat 37° C. and the virus harvest in the perfused cell culture supernatantwas discarded. From the 2nd day after infection, virus replication wascarried out at 33° C. and the perfusion rate of 2 fermenter volumes/daywas reduced to 0 within 7 days. The trypsin necessary for virusreplication was present in the UltraCHO medium which was used for theperfusion in a concentration of 10 μg/ml. The virus harvest (=perfusedcell culture supernatant) was collected at 4° C. and the virusreplication over 7 days was determined as the amount of antigen (TableVIIIb).

TABLE VIIIa Replication of MDCK 33016 cells in the perfusion fermenterLive Total cell count cell count Perfusion Day [10⁵/ml] [10⁵/ml] [1/day]0 0.6 0.6 0 3 8.0 8.3 0 4 14.6 17.5 1.1 5 33.3 34.7 1.1 6 49.8 53.6 2.17 84.5 85.6 3.9 9 82.6 84.9 4.0 9 100.8 104.8 4.1 10 148.5 151.0 4.0 11175.8 179.6 3.9

TABLE VIIIb Replication of influenza A/PR/8/34 in the perfusionfermenter (cell line MDCK 33016), measured as antigen content (HA units)in the cumulated perfused cell culture supernatant Day after HA contentin virus Medium addition Total amount virus infection harvest(perfusion) harvest 1 <4 41 01 2 8 41 41 3 64 31 71 4 256 21 91 5 204821 111 6 4096 21 121 7 4096 01 121

EXAMPLE 10 Preparation of an Experimental Influenza Vaccine

An experimental vaccine was prepared from influenza virus A/PR/8/34 fromExample 2—A/PR/8 replicated at 37° C.—(Experiment 2; vaccine A) andExample 4—A/PR/8 replicated at 33° C.—(vaccine B). The influenza Virusesin the cell culture medium were separated from cells and cell fragmentsby low-speed centrifugation (2000 g, 20 min, 4° C.) and purified by asucrose gradient centrifugation (10 to 50% (wt/wt) of linear sucrosegradient, 30,000 g, 2 h, 4° C.). The influenza virus-containing band wasobtained, diluted 1:10 with PBS pH 7.2, and sedimented at 20,000 rpm,and the pellet was taken up in PBS (volume 50% of the original cellculture medium). The influenza viruses were inactivated withformaldehyde (addition twice of 0.025% of a 35% strength formaldehydesolution at an interval of 24 h, incubation at 20° C. with stirring). 10NMRI mice each, 18 to 20 g in weight, were inoculated with 0.3 ml eachof these inactivated experimental vaccines on day 0 and day 28 bysubcutaneous injection. 2 and 4 weeks after the inoculation and also 1and 2 weeks after revaccination, blood was taken from the animals todetermine the titer of neutralizing antibodies against A/PR/8/34. Todetermine the protection rate, the mice were exposed 2 weeks afterrevaccination (6 weeks after the start of the experiment) by intranasaladministration of 1000 LD₅₀ (lethal dose 50%). The results of theexperiment are compiled in Table IX.

TABLE IX Efficacy of experimental vaccines: for vaccine A the influenzavirus A/PR/8/34 was replicated at 37° C. and for vaccine B at 33° C. Thetiter of neutralizing antibodies against A/PR/8 and also the protectionrate after exposure of the mice were investigated Protection Titer ofneutralizing antibodies/ml* rate 2 w Number pvacc 4 w pvacc 1 w prevacc2 w prevacc living/total Vaccine A <28 56 676 1,620 1/10 Vaccine B 1121,549 44,670 112,200 9/10 *Weeks after vaccination (w pvacc) and weeksafter revaccination (w prevacc)

The experiments confirm that influenza viruses which had been replicatedat 37° C. in cell culture with a high antigen yield (HA titer) onlyinduced a low neutralizing antibody titer in the mouse and barelyprovided protection, while influenza viruses which had been replicatedat 33° C. in cell culture also with a high antigen yield (HA titer)induced very high neutralizing antibody titers in the mouse and led tovery good protection.

1. A process for making a vaccine for administration to humans oranimals, the process comprising: (a) incubating MDCK cells in aserum-free medium; (b) adding influenza viruses to the serum-free mediumto infect the MDCK cells incubated in step (a), wherein protease ispresent in the serum-free medium or added to the serum-free mediumbefore, during, or after infection with the influenza viruses; (c)culturing the MDCK cells infected in step (b) to replicate the influenzaviruses; (d) isolating the influenza viruses replicated in step (c); and(e) formulating the influenza viruses isolated in step (d) to providethe vaccine, wherein the vaccine contains disintegrated viruses.
 2. Theprocess of claim 1, wherein the MDCK cells incubated in step (a) areincubated in a perfusion system.
 3. The process of claim 1, wherein theMDCK cells incubated in step (a) are incubated in a batch process. 4.The process of claim 1, wherein the MDCK cells infected in step (b) arecultured in step (c) at a regulated pH and pO₂.
 5. The process of claim1, wherein the serum-free medium is a protein-free medium.
 6. Theprocess of claim 1, wherein the influenza viruses replicated in step (c)are isolated in step (d) by a process comprising centrifugation.
 7. Theprocess of claim 6, wherein the influenza viruses replicated in step (c)are isolated in step (d) by a process comprising sucrose gradientcentrifugation.
 8. The process of claim 1, wherein the MDCK cells are ofthe cell line MDCK 33016 (DSM ACC 2219).
 9. The process of claim 1,wherein the influenza viruses isolated in step (d) are inactivated byformaldehyde.
 10. The process of claim 1, wherein the vaccine comprisesan adjuvant.
 11. The process of claim 1, wherein the influenza virusesisolated in step (d) are disintegrated using a detergent, prior to step(e).
 12. The process of claim 1, wherein the influenza viruses isolatedin step (d) are disintegrated using a solvent, prior to step (e). 13.The process of claim 1, wherein the influenza viruses isolated in step(d) are disintegrated using a detergent and a solvent, prior to step(e).
 14. The process of claim 1, wherein the vaccine is a subunitvaccine.
 15. The process of claim 1, wherein step (e) comprisesadjusting the vaccine containing disintegrated viruses to a desiredamount of antigen.
 16. The process of claim 1, wherein the protease istrypsin and the trypsin is present in the serum-free medium, or added tothe serum-free medium before, during, or after infection with influenzaviruses, at a concentration from 1 to 200 μg/ml.
 17. The process ofclaim 1, wherein the influenza viruses have a genome segment from strainA/PR/8134.
 18. The process of claim 1, wherein the MDCK cells areincubated adherently.
 19. The process of claim 18, wherein the MDCKcells are incubated adherently on microcarriers.
 20. The process ofclaim 1, wherein the MDCK cells are incubated in suspension.
 21. Theprocess of claim 1, wherein the MDCK cells infected in step (b) arecultured in step (c) at a temperature below 37° C.
 22. The process ofclaim 21, wherein the MDCK cells infected in step (b) are cultured instep (c) at a temperature of 30° C. to 36° C.
 23. The process of claim21, wherein the MDCK cells infected in step (b) are cultured in step (c)for 2 to 10 days.
 24. The process of claim 1, wherein the process isperformed in a culture vessel that is not opened after adding theinfluenza viruses in step (b) or before isolating the influenza virusesin step (d).
 25. The process of claim 1, wherein (i) the influenzaviruses replicated in step (c) are isolated in step (d) by a processcomprising centrifugation, (ii) the influenza viruses isolated in step(d) are inactivated by formaldehyde, and (iii) the MDCK cells areincubated adherently.
 26. A process for making a vaccine foradministration to humans or animals, the process comprising: (a)incubating MDCK cells which can be infected by influenza viruses in aserum-free medium; (b) adding the influenza viruses to the serum-freemedium to infect the MDCK cells incubated in step (a), wherein proteaseis present in the serum-free medium or added to the serum-free mediumbefore, during, or after infection with influenza viruses; (c) culturingthe MDCK cells infected in step (b) to replicate the influenza viruses;(d) isolating the influenza viruses replicated in step (c) by a processcomprising centrifugation; and (e) formulating the influenza virusesreplicated in step (c) to provide the vaccine, wherein the vaccinecontains live attenuated viruses.
 27. The process of claim 26, whereinstep (e) comprises adjusting the influenza viruses isolated in step (d)to a desired titer.
 28. The process of claim 26, wherein step (e)further comprises lyophilizing the viruses.
 29. The process of claim 26,wherein step (e) further comprises stabilizing the viruses in liquidform.
 30. A process for making a vaccine for administration to humans oranimals, the process comprising: (a) incubating MDCK cells adherently ina serum-free medium; (b) adding influenza viruses, having a genomesegment from strain A/PR/8/34, to the serum-free medium to infect theMDCK cells incubated in step (a), wherein protease is present in theserum-free medium or added to the serum-free medium before, during, orafter infection with the influenza viruses; (c) culturing the MDCK cellsinfected in step (b) to replicate the influenza viruses; (d) isolatingthe influenza viruses replicated in step (c); (e) inactivating theinfluenza viruses isolated in step (d) by formaldehyde; and (f)formulating the influenza viruses inactivated in step (e) to provide thevaccine, wherein the vaccine contains disintegrated viruses.